This application relates to hepatocyte growth factor receptor antagonists. The application also relates to the use of the antagonists in therapy or diagnosis of particular pathological conditions in mammals, including cancer.
Hepatocyte growth factor (xe2x80x9cHGFxe2x80x9d) functions as a growth factor for particular tissues and cell types. HGF was identified initially as a mitogen for hepatocytes [Michalopoulos et al., Cancer Res., 44:4414-4419 (1984); Rusasel et al., J. Cell. Physiol., 119:183-192 (1984); Nakamura et al., Biochem. Biophys. Res. Comm., 122:1450-1459 (1984)]. Nakamura et al., supra. reported the purification of HGF from the serum of partially hepatectomized rates. Subsequently, HGF was purified from rat platelets, and its subunit structure was determined [Nakamura et al., Proc. Natl. Acad. Sci. USA, 83:6489-6493 91986); Nakamura et al., FEBS Letters, 224:311-316 (1987)]. The purification of human HGF (xe2x80x9chuHGFxe2x80x9d) from human plasma was first described by Gohda et al., J. Clin. Invest. 81:414-419 (1988).
Both rat HGF and huHGF have been molecularly cloned, including the cloning and sequencing of a naturally occurring variant lacking 5 amino acids designated xe2x80x9cdelta5 HGFxe2x80x9d [Miyazawa et al., Biochem, Biophys. Res. Comm., 163:967-973 (1989); Nakamura et al., Nature 342:440-443 (1989); Seki et al., Biochem. Biophys. Res. Commun., 172:321-327 (1990); Tashiro et al., Proc. Natl. Acad. Sci. USA, 87:3200-3204 (1990); Okajima et al., Eur. J. Biochem., 193:375-381 (1990)].
The mature form of huHGF, corresponding to the major form purified from humam serum, is a disulfide linked heterodimer derived by proteolytic cleavage of the human pro-hormone between amino acids R494 and V495. This cleavage process generates a molecule composed of an xcex1-subunit of 440 amino acids (Mr 69 kDa) and a xcex2-subunit of 234 amino acids (Mr 34 kDa). The nucleotide sequence of the huHGF cDNA reveals that both the xcex1- and the xcex2-chains are contained in a single open reading frame coding for a pre-pro precursor protein. In the predicted primary structure of mature huHGF, an interchain Sxe2x80x94S bridge is formed between Cys 487 of the xcex1-chain and Cys 604 in the xcex2-chain [see Nakamura et al., Nature, supra]. The N-terminus of the xcex1-chain is preceded by 54 amino acids, starting with a methionine group. This segment includes a characteristic hydrophobic leader (signal) sequence of 31 residues and the prosequence. The xcex1-chain starts at amino acid (aa) 55, and contains four kringle domains. The kringle 1 domain extends from about aa 128 to about aa 206, the kringle 2 domain is between about as 211 and about as 288, the kringle 3 domain is defined as extending from about aa 303 to about aa 383, and the kringle 4 domain extends from about aa 391 to about aa 464 of the xcex1-chain.
The definition of the various kringle domains is based on their homology with kringle-like domains of other proteins (such as prothrombin and plasminogen), therefore, the above limits are only approximate. To data, the function of these kringles has not been determined. The xcex2-chain of huHGF shows high homology to the catalytic domain of serine proteases (38% homology to the plasminogen serine protease domain). However, two of the three residues which form the catalytic triad of serine proteases are not conserved in huHGF. Therefore, despite its serine protease-like domain, huHGF appears to have no proteolytic activity. and the precise role of the xcex2-chain remains unknown. HGF contains four putative glycosylation sites, which are located at positions 294 and 402 of the xcex1-chain and at positions 566 and 653 of the xcex2-chain.
Comparisons of the amino acid sequence of rat HGF with that of huHGF have revealed that the two sequences are highly conserved and have the same characteristic structural features. The length of the four kringle domains in rat HGF is exactly the same as in huHGF. Furthermore, the cysteine residues are located in exactly the same positions, an indication of similar three-dimensional structures [Okajima et al., supra; Tashiro et al., supra].
In a portion of cDNA isolated from human leukocytes, in-frame deletion of 15 base pairs was observed. Transient expression of the cDNA sequence in COS-1 cells revealed that the encoded HGF molecule (delta5 HGF) lacking 5 amino acids in the kringle 1 domain was fully functional [Seki et al., supra].
A naturally occurring huHGF variant has been identified which corresponds to an alternative spliced form of the huHGF transcript containing the coding sequences for the N-terminal finger and first two kringle domains of mature huHGF [Chan et al., Science, 254:1382-1385 (1991); Miyazawa et al., Eur. J. Biochem., 197:15-22 (1991)]. This variant, designated HGF/NK2, has been proposed by some investigators to be a competitive antagonist of mature huHGF. Hartmann et al. have reported, however, that HGF/NK2 may retain the ability to cause MDCK cells to scatter [Hartmann et al., Proc. Natl. Acad. Sci., 89:11574-11578 (1992)].
Another HGF variant, designated HGF/NK1, has also been reported to act as a competitive antagonist of HGF [Lokker et al., J. Biol. Chem., 268:17145-17150 (1993); Lokker et al., EMBO J., 11:2503-2510 (1992)]. That HGF/NK1 molecule, containing the N-terminal hairpin and the first kringle domain, was found to block binding of HGF to the HGF receptor on A549 human lung carcinoma cells. It was also found, however, that certain concentrations of the HGF/NK1 induced a detectable increase in receptor tyrosine phosphorylation in the A549 cells, suggesting some agonistic activity. Accordingly, it is believed that the agonist or antagonist action of HGF/NK1 may be dependent upon cell type.
HGF and HGF variants are described further in U.S. Pat. Nos. 5,237,158, 5,316,921, and 5,328,837.
A high affinity receptor for HGF has been identified as the product of the c-Met protooncogene [Bottaro et al., Science, 251:802-804 (1991); Naldini et al., Oncogene, 6:501-504 (1991); WO 92/13097 published Aug. 6, 1992; WO 93/15754 published Aug. 19, 1993]. This receptor is usually referred to as xe2x80x9cc-Metxe2x80x9d or xe2x80x9cp190METxe2x80x9d and typically comprises, in its native form, a 190-kDa heterodimeric (a disulfide-linked 50-kDa xcex1-chain and a 145-kDa xcex2-chain) membrane-spanning tyrosine kinase protein [Park et al., Proc. Natl. Acad. Sci. USA, 84:6379-6383 (1987)]. Several truncated forms of the c-Met receptor have also been described [WO 92/20792; Prat et al., Mol. Cell. Biol., 11:5954-5962 (1991)].
The binding activity of HGF to c-Met is believed to be conveyed by a functional domain located in the N-terminal portion of the HGF molecule, including the first two kringles [Matsumoto et al., Biochem. Biophys. Res. Commun., 181:691-699 (1991); Hartmann et al., Proc. Natl. Acad. Sci., 89:11574-11578 (1992); Lokker et al., EMBO J., 11:2503-2510 (1992); Lokker and Godowski, J. Biol. Chem., 268:17145-17150 (1991)]. The c-Met protein becomes phosphorylated on tyrosine residues of the 145 kDa xcex2-subunit upon HGF binding.
Certain antibodies to this HGFG receptor have been reported in the literature. Several such antibodies are described below.
Prat et al., Mol. Cell. Biol., supra. describe several monoclonal antibodies specific for the extracellular domain the xcex2-chain encoded by the c-Met gene [see also, WO 92/20792]. The monoclonal antibodies were selected following immunization of Balb/c mice with whole living GTL-16 cells (human gastric carcinoma cell line) overexpressing the Met protein. The spleen cells obtained from the immunized mice were fused with Ag8.653 myeloma cells, and hybrid supernatants were screened for binding to GTL-16 cells. Four monoclonal antibodies, referred to as DL-21, DN-30, DN-31 and DO-24, were selected.
Prat et al., Int. J. Cancer, 49:323-328 (1991) describe using anti-c-Met monoclonal antibody DO-24 for detecting distribution of the c-Met protein in human normal and neoplastic tissues [see, also, Yamada et al., Brain Research, 637:308-312 (1994)]. The murine monoclonal antibody DO-24 was reported to be an IgG2a isotype antibody.
Crepaldi et al., J. Cell Biol., 125:313-320 (1994) report using monoclonal antibodies DO-24 and DN-30 [described in Prat et al., Mol. Cell. Biol., supra] and monoclonal antibody DQ-13 to identify subcellular distribution of HGF receptors in epithelial tissues and in MDCK cell monolayers. According to Crepaldi et al., monoclonal antibody DQ-13 was raised against a peptide corresponding to nineteen COOH-terminal amino acids (from Ser1372 to Ser1390) of the human c-Met sequence.
A monoclonal antibody specific for the cytoplasmic domain of human c-Met has also been described [Bottaro et al., supra].
Several of the monoclonal antibodies referenced above are commercially available from Upstate Biotechnology Incorporated, Lake Placid, N.Y. Monoclonal antibodies DO-24 and DL-21, specific for the extracellular epitope of c-Met, are available from Upstate Biotechnology Incorporated. Monoclonal antibody DQ-13, specific for the intracellular epitope of c-Met, is also available from Upstate Biotechnology Incorporated.
In addition to binding c-Met, it is recognized that HGF binds to some heparin and heparan sulfate proteoglycans which are present on cell surfaces or in extracellular matrices [Rouslahti et al., Cell. 64:867-869 (1991); Lyon et al., J. Biol. Chem., 269:11216-11223 (1994)]. Heparan sulfate is a glycosaminoglycan similar in composition and structure to heparin and is found on many mammalian cell surfaces. Various hypotheses have been proposed to explain the role of heparain and heparan sulfate proteoglycans xe2x80x9c(HSPGs)xe2x80x9d in the regulation of certain growth factor activity. For example, it has been hypothesized that upon binding heparin or HSPGs, certain growth factors may have a more favorable conformation for binding to their respective high affinity receptors [Lindahl et al., Annual Rev. Biochem., 47:385-417 (1995)]; that HSPGs may serve as docking sites for certain growth factors facilitating the presentation of ligand to its high affinity receptor [Yayon et al., Cell, 64:841-848 (1991); Moscatelli et al., J. Biol. Chem., 267:25803-25809 (1992); Nugent et al., Biochemistry, 31:8876-8883 (1992)]; and that HSPGs may promote ligand dimerization facilitating receptor activation [Ornitz et al., Mol. Cell. Biol., 12:240-247 (1992); Spivak-Kroizman et al., Cell, 79:1015-1024 (1994)]. It has further been postulated that certain growth factors are more stable or resistant to proteolytic activity [Damon et al., J. Cell. Physiol., 138:221-226 (1989); Mueller et al., J. Cell. Physiol., 140:439-448 (1989); Rosengart et al., Biochem. Biophys. Res. Commun., 152:432-440 (1998)] and denaturation [Copeland et al., Arch. Biochem. Biophys., 289:53-61 (1994)] when bound to heparin. Coincubation of HGF with soluble heparin and other heparin-like molecules has been reported to promote dimerization/oligomerization of HGF and to potentiate HGF mitogenic activity, [see e.g., WO 94/09969 published Mar. 16, 1995; Zioncheck et al., J. Biol. Chem., 270:16871-16878 91995)].
Mizuno et al. describe some experiments which attempted to locate heparin-binding sites within the HGF molecule [Mizuno et al., J. Biol. Chem., 269:1131-1136 (1994)]. Mizuno et al. constructed variously deleted mutant HGFs [d-K1 (deletion of first kringle domain); d-K2 (deletion of second kringle domain); d-K3 (deletion of third kringle domain); d-K4 (deletion of fourth kringle domain); d-beta (deletion of beta chain); d-H (deletion of N-terminal hairpin loop); and HK1K2 (consisting of N-terminal hairpin loop and the first and second kringle domains)] and examined their respective binding to an immobilized heparin column. The reference reports that the d-H and d-KJ2 mutants exhibited decreased binding to heparin affinity columns, while the native HGF and the other constructed HGF mutants tightly bound to the heparin columns.
Various biological activities have been described for HGF and its c-Met receptor [see, generally, Chan et al., Hepatocyte Growth Factor-Scatter Factor (HGF-SF) and the C-Met Receptor, Goldberg and Rosen, eds., Birkhauser Verlag-Basel (1993), pp. 67-79]. It has been observed that levels of HGF increase in the plasma of patients with hepatic failure [Gohda et al., supra] and in the plasma [Lindroos et al., Hepatol., 13:734-750 (1991)] or serum [Asami et al., J. Biochem., 109:8-13 (1991)] of animals with experimentally induced liver damage. The kinetics of this response are usually rapid, and precedes the first round of DNA synthesis during liver regeneration. HGF has also been shown to be a mitogen for certain cell types, including melanocytes, renal tubular cells, keratinocytes, certain endothelial cells and cells of epithelial origin [Matsumoto et al., Biochem. Biophys. Res. Commun., 176:45-51 (1991); Igawa et al., Biochem. Biophys. Res. Commun., 174:831-838 91991); Han et al., Biochem., 30:9768-9780 (1991); Rubin et al., Proc. Natl. Acad. Sci. USA, 88:415-419 (1991)]. Both HGF and the c-Met protooncogene have been postulated to play a role in microglial reactions to CNS injuries [DiRenzo et al., Oncogene, 8:219-222 (1993)].
HGF can also act as a xe2x80x9cscatter factorxe2x80x9d, an activity that promotes the dissociation of epithelial and vascular endothelial cells in vitro [Stroker et al., Nature, 327:239-242 (1987); Weidner et al., J. Cell Biol., 111:2097-2108 (1990); Naldini et al., EMBO J., 10:2867-2878 (1991); Giordano et al., Proc. Natl. Acad. Sci. USA, 90:649-653 (1993)]. Moreover, HGF has recently been described as an epithelial morphogen [Montesano et al., Cell, 67:901-908 (1991)]. Therefore, HGF has been postulated to be important in tumor invasion [Comoglio, Hepatocyte Growth Factor-Scatter Factor (HGF-SF) and the C-Met Receptor, Goldberg and Rosen, eds., Birkhauser Verlag-Basel (1993), pp. 131-165]. Bellusci et al., Oncogene, 9:1091-1099 (1994) report that HGF can promote motility and invasive properties of NBT-11 bladder carcinoma cells.
c-Met RNA has been detected in several murine myeloid progenitor tumor cell lines [Iyer et al., Cell Growth and Differentiation, 1:87-95 (1990)]. Further, c-Met is expressed in various human solid tumors [Prat et al., Int. J. Cancer, supra]. Overexpression of the c-Met oncogene has also been suggested to play a role in the pathogenesis and progression of thyroid tumors derived from follicular epithelium [DiRenzo et al., Oncogene, 7:2549-2553 (1992)]. Chronic c-Met/HGF receptor activation has also been observed in certain malignancies [Cooper et al., EMBO J., 5:2623 (1986); Giordano et al., Nature, 339:155 (1989)].
In view of the role of HGF and/or c-Met in potentiating or promoting such diseases or pathological conditions, it would be useful to have a means of substantially reducing or inhibiting one or more of the biological effects of HGF and c-Met.
The intention provides HGF receptor antagonists which are capable of specifically binding to a HGF receptor. Preferred HGF receptor antagonists are capable of substantially reducing or inhibiting the mitogenic, motogenic (migration or scatter) or other biological activity of HGF or HGF receptor activation, and thus are useful in the treatment of various diseases and pathological conditions such as cancer. In one embodiment of the invention, the HGF receptor antagonists is an antibody. Preferably, the antagonist is a monoclonal antibody, and more preferably, is a Fab fragment of a monoclonal antibody.
The invention also provides hybridoma cell lines which produce HGF receptor antagonist monoclonal antibodies.
The invention also provides HGF receptor antagonists that comprise isolated polypeptide comprising the amino acid sequences of FIG. 1A (SEQ ID NO:1) and FIG. 1B (SEQ ID NO:2). The polypeptides consisting of the amino acid sequences of FIG. 1A (SEQ ID NO:1) and FIG. 1B (SEQ ID NO:2) correspond to the light chain and heavy chain, respectively, of monoclonal antibody 5D5 Fab, described herein.
The invention also provides chimeric molecules comprising HGF receptor antagonist linked or fused to another, heterologous polypeptide or polymer. An example of such a chimeric molecule comprises a HGF receptor antagonist amino acid sequence linked or fused to an albumin sequence or polyethylene glycol (xe2x80x9cPEGxe2x80x9d) sequence.
The invention further provides an isolated nucleic acid molecule encoding HGF receptor antagonist. In one aspect, the nucleic acid molecule is RNA or DNA that encodes a HGF receptor antagonist or is complementary to a nucleic acid sequence encoding such HGF receptor antagonist, and remains stably bound to it under stringent conditions. In one embodiment, the nucleic acid sequences are selected from:
(a) the nucleic acid sequence of FIG. 1A that codes for residue 1 to residue 220 (i.e., nucleotides 1 through 660; SEQ ID NO:3), inclusive;
(b) the nucleic acid sequence of FIG. 1B that codes for residue 1 to residue 230 (i.e., nucleotides 1 through 690; SEQ ID NO:4), inclusive; or
(c) a nucleic acid sequence corresponding to the sequence of (a) or (b) within the scope of degeneracy of the genetic code.
The invention also provides a replicable vector comprising the nucleic acid molecule(s) encoding the HGF receptor antagonist operably linked to control sequence(s) recognized by a host cell transfected or transformed with the vector. A host cell comprising the vector or the nucleic acid molecule(s) is also provided. A method of producing HGF receptor antagonist which comprises culturing a host cell comprising the nucleic acid molecule(s) and recovering the protein from the host cell culture is further provided.
The invention also provides pharmaceutical compositions comprising one or more HGF receptor antagonists in a pharmaceutically-acceptable carrier. In one embodiment, the pharmaceutical composition may be included in an article of manufacture or kit.
The invention also provides methods of employing HGF receptor antagonists, including methods of inhibiting HGF receptor activation.
The invention further provides methods for treating cancer comprising administering to a mammal diagnosed as having cancer an effective amount of a HGF receptor antagonist. The HGF receptor antagonist alone may be administered to the mammal, or alternatively, may be administered to the mammal in combination with other therapeutic agents such as anti-cancer agents.
It is believed that the antagonists can be used to block binding of HGF to HGF receptor(s) or substantially prevent HGF receptor activation, thereby treating pathologic conditions associated with binding of HGF to HGF receptor(s) or with the activation of HGF receptor(s).